1. Field of the Invention
The present invention relates generally to the field of plant transformation. More particularly, it concerns methods for reducing the transgene copy number of transformants.
2. Description of Related Art
Recent advances in molecular biology have dramatically expanded the ability of scientists to manipulate the germplasm of animals and plants. Genes controlling specific phenotypes, for example, specific polypeptides that lend insect, antibiotic and herbicide resistance, have been identified in certain germplasm and isolated therefrom. Even more important has been the ability to introduce these genes into a heterologous organism. This transformation may be accomplished even where the recipient organism is from a different phylum, genus or species from that which donated the gene (heterologous transformation).
Many attempts have been made to genetically engineer desired traits into plant genomes by introduction of exogenous genes using genetic engineering techniques. The uptake of new DNA by recipient plant cells has been accomplished by various means, including Agrobacterium infection (Nester et al., 1984), polyethylene glycol (PEG)-mediated DNA uptake (Lorz et al., 1985), electroporation of protoplasts (Fromm et al., 1986) and microprojectile bombardment (Klein et al., 1987).
Microprojectile bombardment is a particularly advantageous transformation technique in that, in addition to being an effective means of reproducibly stably transforming monocots, neither the isolation of protoplasts (Cristou et al., 1988) nor the susceptibility to Agrobacterium infection is required (see, for example, Gordon-Kamm et al., 1990). While successful recovery of fertile transgenic plants has been accomplished in many instances by microprojectile bombardment (Fromm et al., 1990; Gordon-Kamm et al., 1990; Walters et al., 1992), efforts have continued to optimize the efficiency of transformation. Parameters which have been suggested to be important include particle size relative to the recipient cell, particle number per cell, the quantity and timing of DNA delivery, how the cells involved in transformation react to DNA particle bombardment and limitation of physical trauma to those cells (Klein et al., 1988a. 1988b.; Russell et al., 1992; Vain et al., 1993; Hunold et al., 1994).
One difficulty associated with microprojectile bombardment transformation and other transformation systems has been the relative inability to control the copy number of introduced transgenes. Specifically, using previous transformation techniques, many transformants are produced with multiple transgene copies. High to moderate copy number transformation events are non-desirable from a number of standpoints. For example, multiple-copies of a transgene can cause co-suppression, thereby "turning off" expression of the transgene. Even in the absence of co-suppression, expression may be limited and may vary over generations. Additionally, recombination can occur between multiple gene copies leading to rearrangements in the transgenes. Complex transformation events may also exhibit non-Mendelian inheritance, greatly complicating efforts to breed transformants.
Even using traditional transformation techniques, some single copy number transformants are produced, but the relative proportion of these is typically low. When using microprojectile bombardment, frequently less than 5% of the transformants recovered posses a single transgene copy. Therefore, in order to obtain the desired single copy transformants, current technology necessitates the screening of large numbers of transformants using labor-intensive screening techniques such as Southern hybridization. This screening represents a significant burden upon scientists, and generally slows efforts in the production of novel transformants having value to the consumer. There is, therefore, a great need in the art to identify specific methods and compositions which may be used to efficiently produce low-copy transformants.